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Transforming growth factor ␤ (TGF-␤) ligands exert their biological effects through type II (T␤RII) and type I receptors (T␤RI). Unlike TGF-␤1 and -␤3, TGF-␤2 appears to require the co-receptor betaglycan (type III receptor, T␤RIII) for high affinity binding and signaling. Recently, the T␤RIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-␤2 null mouse, implying the existence of T␤RIII independent mechanisms for TGF-␤2 signaling. Because a variant of the type II receptor, the type II-B receptor (T␤RII-B), has been suggested to mediate TGF-␤2 signaling in the absence of T␤RIII, we directly tested the ability of T␤RII-B to bind TGF-␤2. Here we show that the soluble extracellular domain of the type II-B receptor (sT␤RII-B.Fc) bound TGF-␤1 and TGF-␤3 with high affinity (K d values ‫؍‬ 31.7 ؎ 22.8 and 74.6 ؎ 15.8 pM, respectively), but TGF-␤2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sT␤RII.Fc). However, sT␤RII.Fc or sT␤RII-B.Fc in combination with soluble type I receptor (sT␤RI.Fc) formed a high affinity complex that bound TGF-␤2, and this complex inhibited TGF-␤2 in a biological inhibition assay. These results show that TGF-␤2 has the potential to signal in the absence of T␤RIII when sufficient TGF-␤2, T␤RI, and T␤RII or T␤RII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-␤ receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.Transforming growth factor-␤ (TGF-␤) 1 represents a large superfamily of dimeric growth factors that include the TGF-␤s, inhibins, activins, Mullerian inhibiting substance, growth and differentiation factors, and bone morphogenetic proteins (BMPs) in mammals (1, 2). These cytokines play important roles in an array of processes such as growth, differentiation, and development (3). There are three TGF-␤ isoforms that share a high degree of homology and overlapping biological activities (4). However, distinct expression patterns and unique, isoform-specific phenotypes of the corresponding knockout mice demonstrate significant non-redundancy of TGF-␤ function (5-9).TGF-␤s exert their biological effects through three cell surface receptors designated as type I, II, and III (T␤RI, T␤RII, and T␤RIII) (2), all of which have been cloned (10 -13). In addition, the type II-B receptor (T␤RII-B), an alternatively spliced isoform of T␤RII containing an insert of 26 amino acids replacing Val 51 , has also been identified (14 -16). Type I and type II receptors have intracellular serine/threonine kinase domains, whereas the type III receptor has only a short intracellular domain. On binding of TGF-␤ ligands, constitutively active type II receptors recruit and phosphorylate type I receptors; the activated type I receptor kinase then interacts with and phosphorylates downstream signaling ...

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.